Variola (smallpox) virus is among the most deadly potential agents of biological warfare and bioterrorism. Treatment options are very limited. The current smallpox vaccine-vaccinia virus-induces serious side effects. Knowledge of vaccinia virus proteins that both suppress immunity and contribute to pathogenesis may lead to therapies for poxviruses and to safer, more effective vaccines. The variola and vaccinia viruses encode proteins that inhibit innate immune signaling, such as Toll-like Receptor (TLR) signaling. The TLRs control innate (and thereby adaptive) immune responses by detecting a wide variety of microbial pathogens. Once engaged by a pathogen, the TLRs stimulate intracellular pathways that ultimately activate NF-?B and IRF3 transcription factors, which drive expression of cytokines, chemokines, interferons, and co-stimulatory molecules. RIG-I is a member of another family of innate immune receptors involved in the recognition of intracellular pathogens, in particular cytoplasmic dsRNA. RIG-I activates NF-?B and IRF3 signaling, similar to the TLRs. Poxviruses encode several inhibitors of innate immune signaling, and they are critical for pathogenesis. For example, N1L inhibits both NF-?B and IRF3 activation following TLR stimulation, thereby suppressing production of TNF and Type I interferons. N1L is known to physically target the IKK complex, which is required for IRF3 and NF-?B activation. Loss of N1L creates vaccinia virus with 10,000-fold reduced virulence. This grant is focused on understanding in molecular detail the mechanisms by which N1L inhibits TLR and RIG-I signaling and how this innate immune inhibition contributes to pathogenesis. In addition, we have identified in vaccinia virus another N1L family member, C16L, which inhibits TLR3 signaling via an unknown mechanism. We propose to investigate this new innate immune inhibitor, probing its mechanism of action and its role in viral virulence. Together, these studies will advance our long-term goal to characterize the mechanisms involved in viral subversion of signal transduction that contribute to viral pathogenesis. Public Health Relevance: The re-emergence of smallpox (and related poxviruses) poses a serious public health threat. This study will examine poxviral factors that contribute to death and disease by interfering with immune responses. A deeper understanding of these processes may lead to new anti-viral therapies.